CN104673706A - 苏云金芽胞杆菌fh21、杀虫基因,表达蛋白及其应用 - Google Patents
苏云金芽胞杆菌fh21、杀虫基因,表达蛋白及其应用 Download PDFInfo
- Publication number
- CN104673706A CN104673706A CN201410798930.6A CN201410798930A CN104673706A CN 104673706 A CN104673706 A CN 104673706A CN 201410798930 A CN201410798930 A CN 201410798930A CN 104673706 A CN104673706 A CN 104673706A
- Authority
- CN
- China
- Prior art keywords
- gene
- cry1ja3
- cry1id3
- cry1bb3
- cry1da4
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 76
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 18
- 241000193388 Bacillus thuringiensis Species 0.000 title claims abstract description 14
- 229940097012 bacillus thuringiensis Drugs 0.000 title claims abstract description 14
- 230000014509 gene expression Effects 0.000 title claims abstract description 10
- 239000002917 insecticide Substances 0.000 title abstract description 7
- 241000607479 Yersinia pestis Species 0.000 claims abstract description 25
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- 230000001580 bacterial effect Effects 0.000 claims description 22
- 230000000749 insecticidal effect Effects 0.000 claims description 16
- 235000018102 proteins Nutrition 0.000 claims description 13
- 241000500437 Plutella xylostella Species 0.000 claims description 9
- 230000002147 killing effect Effects 0.000 claims description 9
- 230000000813 microbial effect Effects 0.000 claims description 7
- 239000000575 pesticide Substances 0.000 claims description 7
- 241000255967 Helicoverpa zea Species 0.000 claims description 6
- 241001147398 Ostrinia nubilalis Species 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 6
- 230000002068 genetic effect Effects 0.000 claims description 4
- 239000000470 constituent Substances 0.000 claims description 2
- 235000003869 genetically modified organism Nutrition 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 12
- 230000009261 transgenic effect Effects 0.000 abstract description 8
- 241000255777 Lepidoptera Species 0.000 abstract description 7
- 230000001988 toxicity Effects 0.000 abstract description 5
- 231100000419 toxicity Toxicity 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract 1
- 230000003111 delayed effect Effects 0.000 abstract 1
- 239000003814 drug Substances 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- 231100000086 high toxicity Toxicity 0.000 abstract 1
- 241000238631 Hexapoda Species 0.000 description 18
- 241000196324 Embryophyta Species 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 244000144987 brood Species 0.000 description 10
- 244000037671 genetically modified crops Species 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000004321 preservation Methods 0.000 description 7
- 230000001018 virulence Effects 0.000 description 7
- 239000002158 endotoxin Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000002689 soil Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000256247 Spodoptera exigua Species 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 101100497223 Bacillus thuringiensis cry1Ag gene Proteins 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 3
- 101710151559 Crystal protein Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- HZLHRDBTVSZCBS-UVJJDBRNSA-N 4-[(e)-(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline;hydrochloride Chemical compound Cl.C1=CC(=N)C(C)=C\C1=C(C=1C=C(C)C(N)=CC=1)/C1=CC=C(N)C=C1 HZLHRDBTVSZCBS-UVJJDBRNSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 101100497222 Bacillus thuringiensis cry1Af gene Proteins 0.000 description 2
- 101100007619 Bacillus thuringiensis cry1Ja gene Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 101150041868 cry1Aa gene Proteins 0.000 description 2
- 101150065438 cry1Ab gene Proteins 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000028070 sporulation Effects 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 101100497226 Bacillus thuringiensis cry1Bb gene Proteins 0.000 description 1
- 101100007618 Bacillus thuringiensis cry1Id gene Proteins 0.000 description 1
- 101100497233 Bacillus thuringiensis subsp. aizawai cry1Da gene Proteins 0.000 description 1
- 101100497219 Bacillus thuringiensis subsp. kurstaki cry1Ac gene Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000654838 Exosporium Species 0.000 description 1
- 108700001097 Insect Genes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000193157 Paraclostridium bifermentans Species 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241000270666 Testudines Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000002016 colloidosmotic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229920001940 conductive polymer Polymers 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 101150086784 cry gene Proteins 0.000 description 1
- 101150092571 cry1Ac gene Proteins 0.000 description 1
- 101150008638 cry1Bb gene Proteins 0.000 description 1
- 101150021345 cry1Da gene Proteins 0.000 description 1
- 101150043595 cry1Id gene Proteins 0.000 description 1
- 101150102059 cry3Aa gene Proteins 0.000 description 1
- 101150060198 cyt gene Proteins 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000019371 dormancy process Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000005357 flat glass Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009616 inductively coupled plasma Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000000361 pesticidal effect Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000000352 supercritical drying Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000007669 thermal treatment Methods 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/075—Bacillus thuringiensis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Environmental Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明涉及“苏云金芽胞杆菌FH21、杀虫基因,表达蛋白及其应用”,属于生物防治技术领域。一株苏云金芽胞杆菌菌株FH21,其保藏编号为CGMCC No.10090,从菌株中分离得到四种杀虫蛋白,具有如SEQ ID NO 2、4、6、8所示的氨基酸序列,及编码该杀虫蛋白的基因,优选其的核苷酸序列如SEQ ID NO1、3、5、7所示。菌株和上述基因对鳞翅目害虫具有高毒力,以应用于转化微生物和植物,使之表现出对相关害虫的毒性,并克服、延缓害虫对工程菌和转基因植物的抗药性产生。
Description
技术领域
本发明涉及生物防治技术领域,特别是进一步,本发明涉及对鳞翅目农业害虫具有高毒力的Bt杀虫基因及由该基因所编码的蛋白质。
背景技术
苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是一种分布广泛的革兰氏阳性细菌,是一种对害虫毒力强且对天敌无毒性的昆虫病原微生物,对高等动物和人无毒性。它是目前研究最为深入、使用最为广泛的微生物杀虫剂,对16个目3000多种害虫有活性。Bt在芽胞形成期可形成杀虫晶体蛋白(Insecticidal CrystalProteins,ICPs),也称δ-内毒素(delta-endotoxin),它的形状、结构和大小均与其毒力有着密切关系[Schnepf.E,Crickmore.N,Van Rie.J.,Lereclus.D,Baum.J,Feitelson.J,Zeigler.D.R.,Dean.D.H.Bacillus thuringiensis and its pesticidal crystal proteins.Microbiol.Mol.Biol.Rev,1998,62(3):775-806.]。自1981年Schnepf等克隆了Bt的第一个ICPs基因,并于1985年发表了它的DNA碱基序列及其编码蛋白的氨基酸序列起,目前(2014年4月)共776个,其中cry基因738个,cry模式基因289个;cyt基因38个,cyt模式基因11个。当今,采用喷施化学农药防治手段固然可以减轻害虫对农作物的为害,但化学农药造成环境污染,长期以来,大量喷施化学杀虫剂,不仅会增强害虫的抗药性,使益虫及其它生态区系遭受破坏,而且严重污染环境,提高生产成本,破坏生态平衡。苏云金芽胞杆菌杀虫晶体蛋白因其杀虫效果好、安全、高效等优点而被广泛的应用于虫害防治。苏云金芽胞杆菌除了直接作为生物农药以外,1996年全世界第一例转基因抗虫植物在美国获准应用,它使用的基因来自Bt cry1Ac。在接下来的几年里,转cry1Ab基因的抗虫玉米,转cry3Aa基因的抗虫土豆等相距问世。在中国,自1998年开始正式推广含有cry1Ac/cry1Ab基因的抗虫棉以来,已经被普遍种植。在转基因作物商业化的第一个12年(1996-2007)中,由于能得到持续稳定的收益,农民种植转基因作物量逐年增加。2013年,27个国家的1800万农民种植了1.752亿公顷(4.33亿英亩)的转基因作物,比2012年持续增长了3%,即500万公顷(1200万英亩)。1800万农民从转基因作物中获益,其中90%为资源匮乏的小农户。前五个种植转基因作物的发展中国家是亚洲的中国和印度、拉丁美洲的巴西和阿根廷以及非洲的南非,共种植8270万公顷的转基因作物,占全球转基因作物种植面积的47%,并且这五个国家的人口约占全球70亿人口的41%。中国750万资源匮乏的小农户种植了420万公顷的Bt棉花,采用率为90%,平均每户农 民种植0.5公顷的Bt棉花。转基因作物商业化给工业化国家和发展中国家的农民都带来了经济和环境效益。苏云金芽胞杆菌及其基因发掘已成为可持续发展农业中重要课题。
由于目前商品化的转基因抗虫作物的抗虫基因种类比较单一,如此大面积推广种植存在害虫避难所减少与害虫抗药性上升的风险。因此需要不断分离高毒力的或者新的基因组合来避免害虫抗药性上升的风险。因此,筛选分离克隆新的、高毒力的Bt杀虫基因,可以丰富杀虫基因资源,为转基因作物与工程菌株提供新的基因来源,提高Bt转基因产品的抗虫效果,并且可以降低害虫对Bt毒蛋白的抗性风险,避免新的生态灾难降临,具有重要的经济、社会和生态效益。
发明内容
本发明提供一种对鳞翅目害虫高毒力的苏云金芽胞杆菌菌株FH21,及其杀虫新基因cry1Bb,cry1Da,cry1Id,cry1Ja和其晶体杀虫蛋白,以应用于转化微生物和植物,使之表现出对相关害虫的毒性,并克服、延缓害虫对工程菌和转基因植物的抗药性产生。
苏云金芽胞杆菌菌株FH21,其保藏编号为:CGMCC No.10090。
苏云金芽胞杆菌菌株FH21在杀灭鳞翅目农业害虫中的应用。
杀虫蛋白Cry1Bb3,Cry1Da4,Cry1Id3或Cry1Ja3,其氨基酸序列如SEQ ID NO 2、SEQ ID NO 4、SEQ ID NO6、SEQ ID NO 8所示。
基因cry1Bb3,cry1Da4,cry1Id3或cry1Ja3,编码杀虫蛋白Cry1Bb3,Cry1Da4,Cry1Id3,Cry1Ja3。
所述基因序列如SEQ ID NO 1、SEQ ID NO 3、SEQ ID NO 5、SEQ ID NO 7所示。
一种表达载体,其特征是含有cry1Bb3,cry1Da4,cry1Id3或cry1Ja3基因。
所述表达载体为pEB-cry1Bb3,pEB-cry1Da4,pEB-cry1Id3或pEB-cry1Ja3,其骨架载体为pEB,其结构如图6所示。
一种微生物转化体,其特征是含有cry1Bb3,cry1Da4,cry1Id3或cry1Ja3基因。
基因cry1Bb3,cry1Da4,cry1Id3,cry1Ja3在杀灭鳞翅目农业害虫中的应用。
所述应用是将cry1Bb3,cry1Da4,cry1Id3或cry1Ja3基因表达的蛋白作为生物杀虫剂的有效成分。
本发明人从黑龙江省凤凰山附近土壤中分离得到一株苏云金芽胞杆菌菌株FH21,其保藏编号为CGMCC No.10090,该菌株生物学特性为在生长周期中可以产生芽胞,并且同时产生有毒杀鞘翅目害虫作用的伴胞晶体,其对鳞翅目农业害虫具有很强的杀灭能力;从该菌株中得到4个基因的阳性克隆,即pEB-cry1Bb,pEB-cry1Da,pEB-cry1Id,pEB-cry1Ja(见图6),对其 进行测序分析,在NCBI网站上应用BLAST并应用DNAMAN等生物软件进行分析,分析结果证明所克隆的基因是cry1Bb基因,该基因编码框是由3609个碱基组成,编码氨基酸数量为1203个,经过比对发现该基因所编码的蛋白与Cry1Bb2的氨基酸同源性是最高的,它们的一致性达到了99%,所以该基因应属于第四等级的新基因,提交GenBank获得登录号为KJ619659,经国际δ-内毒素命名委员会正式命名为Cry1Bb3。该蛋白比对结果如下:
>Cry1Bb:
Length=1203
Score=2500bits(6480),Expect=0.0
Identities=1202/1203(99%),Positives=1202/1203(99%),Gaps=0/1203(0%)
在NCBI网站上应用BLAST并应用DNAMAN等生物软件进行分析[[84],分析结果证明所克隆的基因是cry1Da基因,该基因编码框是由3492个碱基组成,编码氨基酸数量为1164个,经过比对发现该基因所编码的蛋白与Cry1Da2的氨基酸同源性是最高的,它们的一致性达到了99%,所以该基因应属于第四等级的新基因,提交GenBank获得登录号为KJ619660,经国际δ-内毒素命名委员会正式命名为Cry1Da4。该蛋白比对结果如下:
>Cry1Da:
Length=1164
Score=2409bits(6242),Expect=0.0
Identities=1160/1164(99%),Positives=1162/1164(99%),Gaps=0/1164(0%)
在NCBI网站上应用BLAST并应用DNAMAN等生物软件进行分析[[84],分析结果证明所克隆的基因是cry1Id基因,该基因编码框是由2160个碱基组成,编码氨基酸数量为720个,经过比对发现该基因所编码的蛋白与Cry1Id1的氨基酸同源性是最高的,它们的一致性达到了99%,所以该基因应属于第四等级的新基因,提交GenBank获得登录号为KJ619661,经国际δ-内毒素命名委员会正式命名为Cry1Id3。该蛋白比对结果如下:
>Cry1Id:
Length=720
Score=1489bits(3856),Expect=0.0
Identities=715/719(99%),Positives=719/719(100%),Gaps=0/719(0%)
测序结果经DNAMAN软件分析处理于NCBI-BLASTP比对,比对结果显示Bt FH21中的cry1类基因编码的杀虫蛋白与cry1Ja的氨基酸同源性最高,一致性为99%。与Cry1Ja1同源性最高仅有5个氨基酸位点存在差异(见表3-7)。基因登录号JQ228425,Bt命名委员会将其命名为cry1Ja3,比对结果如下:
>Cry1Ja
Length=789
Score=2410bits(6246),Expect=0.0
Identities=1161/1166(99%),Positives=1162/1166(99%),,Gaps=0/1166(0%)
cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因可按生物技术的常规方法转化微生物、植物,表现出对相关鳞翅目害虫的毒性。
将上述基因转化菌株,表达得到的蛋白可以制成生物农药用于杀死鳞翅目害虫。同时,可以转入植物构建抗虫转基因植物,用于害虫的防治。
本发明分离克隆的Bt cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因序列及其基因表达产物能够对鳞翅目农业害虫小菜蛾,棉铃虫、玉米螟、甜菜夜蛾产生强毒力,都是很好的生物杀虫基因,有很广泛的应用前景。通过cry1Bb3,cry1Da4,cry1Id3和cry1Ja3可扩大对鳞翅目害虫的杀虫谱。通过应用于转化微生物和植物,使它们表现出对相关害虫的毒性,可克服或延缓昆虫对工程菌和转基因植物抗药性的产生。
菌株保藏信息:
菌种分类命名:苏云金芽孢杆菌(Bacillus thuringiensis)
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏地址:北京市朝阳区北辰西路1号院3号
保藏日期:2014年12月1日
保藏编号:CGMCC No.10090
附图说明
图1光学显微镜下观察菌株FH21菌体的形态,
图2电镜下菌株FH21菌体形态,
图3FH21菌株cry1基因PCR结果,
M:DM2000plus;1:primer L5un4/L3un4;2:primer L5un3/L3un3;3:primer L5un2/L3un2;4:primer L5un1/L3un1,
图4FH21菌株PCR-RFLP结果,
M:DM2000plus;1:RFLP patterns of primer L5un1/L3un1PCR products;2:RFLP patterns of primer L5un3/L3un3PCR products,
图5cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因在大肠杆菌Rosetta(DE3)表达结果,
1:Cry1Ja3precipitate;2:pEB precipitate;3:Cry1Ja3supernatant;4:pEB supernatant 5:pEB supernatant;6:Cry1Id3precipitate;7:Cry1Bb3supernatant;8:Cry1Bb3precipitate;9:Cry1Id3precipitate;11:pEB precipitate;12:pEB empty vector;13:Cry1Da4supernatant;14:Cry1Da4precipitate,
图6cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因表达载体的构建。
具体实施方式
实施例1、分离得到苏云金芽胞杆菌菌株FH21
本申请人的实验室工作人员从黑龙江省凤凰山土壤中分离的得到一株苏云金芽胞杆菌,苏云金芽胞杆菌的芽胞由外向内依次为芽胞外壁、芽胞衣、皮层、芽胞内壁,原生质膜和原生质体。其中皮层的主要成分是肽聚糖,不含营养细胞的多聚糖磷壁酸,它保持着芽胞的脱水状态和耐热性,另一方面,芽胞形成过程中,会产生大量DPA-Ca鳌合物,使得芽胞中的生物大分子形成耐热凝胶,在80℃下热处理20min,苏云金芽胞杆菌芽胞也不会死亡并且休眠的芽胞在75℃的亚致死温度下处理15min,活化效果最好,不但促其快速萌发,还可提高芽胞的成活率(喻子牛1990)。依据这一特性,可实施温度筛选(Knowles B H,Ellar D J.Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis d-endotoxins with different specificity[J].Biochimica et biophysica acta,1987,924:509-518.;戴莲韵,王学聘等.中国八个自然保护区森林土壤中苏云金芽胞杆的分布[J].微生物学报,1994,30(2)117-121)。
1.菌株的分离
1.11)取分装的土样加入到50ml大离心管中,至锥形管锥形处。
2)加灭菌水至15ml处,放入玻璃珠5~10颗。
3)用振荡器将土样打碎。
4)放入水浴锅中80℃,20分钟。
5)取1.5ml的EP管,每个管中加1ml灭菌水,再从50ml管中取10微升菌液加入到EP管中混匀。
6)从EP管中取100微升喷到1/2LB培养基中,涂匀。
7)放入到30℃温箱中培养2~3天。
8)镜检观察。
1.2晶体观察
1.2.1光学显微镜:
将胞晶混合液滴于载玻片上,涂抹均匀,烘干固定,石炭酸复红染液染色3min,清水冲洗,100x油镜进行镜检,石炭酸复红染液配制方法参见文献(Baroy F,Lecadet M M,Deleluse A.Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans[J].Gene,1998,211:293-295)。见图1所示。在1/2LB培养基上培养48 h后形成单菌落,光学显微镜下观察到菌株QZL38菌体为长杆状,芽胞为长圆棒状,晶体为双锥形。
1.2.2电镜显微观察:
扫描电镜制样:孢晶混合液滴于玻璃片上,干燥,经锇酸固定,而后经酒精梯度脱水,临界点干燥,离子溅射喷金(2nm厚),New Bio-TEM H-7500扫描电镜观察拍照。如图2所示。
生物学测定表明,对小菜蛾、甜菜夜蛾的初筛生测结果为校正死亡率为100%。
将该菌株保藏,其保藏编号CGMCC No.10090。
实施例2.获得新基因
21利用cry类基因通用引物检测菌株FH21,引物如下
扩增循环:94℃变性1分钟,56℃退火1分钟,72℃延伸4分钟,25个循环,最后72℃延伸10分钟。
结果如图3所示,菌株FH21进行基因型的PCR鉴定,用cry1类基因鉴定引物获得了大小为不一的PCR产物。
2.2采用快速克隆方法对该菌株中的新sip1A基因进行分离克隆。
用pfuDNA聚合酶,用如下体系进行PCR扩增。
超纯水补至50μL,混匀离心。
扩增循环:94℃变性1分钟,54℃退火1分钟,72℃延伸1分钟,25个循环,最后72℃延伸10分钟。
连接方案
用超纯水补足体积到10μL,充分混匀,16℃连接4h或4℃连接过夜。
设计cry1Bb3,cry1Da4,cry1Id3和cry1Ja3类基因的全长引物,扩增得到全长基因,将其与载体pEB(公开载体,本所实验室有保存,可以对外公开发放)载体进行连接,转化入感受态JM109中,经抗性筛选、PCR鉴定分析,筛选出含有cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因的阳性重组质粒。图4PCR鉴定结果。将纯化片段与载体pEB连接转化大肠杆菌JM109,得到阳性转化子。对插入片断进行测序分析,得到序列SEQ ID NO 1、3、5、7,其氨基酸序列为SEQ ID NO 2、4、6、8所示。
2.3转化方案
2.3.1大肠杆菌转化
1.挑取单菌落于5ml LB震荡培养过夜;
2.按1%接种量接种于LB液体培养基中,37℃,230rpm培养2-2.5hr,(OD600=0.5-0.6);
3.4℃,4,000rpm离心10min;
4.弃上清,加入预冷的0.1M CaCl250ml悬浮细胞,置于冰上30min以上;
5.4℃,4,000rpm离心10min,回收细胞;
6.用2-4ml冰预冷的0.1M CaCl2重悬细胞,分装成200μl/0.5mL离心管中,于4℃保存(可保存一周)。
7.取200μl感受态细胞与5μL连接产物充分混匀,冰浴30min。
8.42℃热激1.5min,冰浴3min。
9.加入800μl LB培养基37℃培养45min。
10.取200μl涂板,加入相应的抗生素,及IPTG,X-gal,37℃培养。
实施例3、基因表达与活性测定
3.1.1在上述克隆提取质粒DNA,转入受体菌Rosetta(DE3)中,获得表达菌株。
IPTG诱导表达后,进行SDS-PAGE蛋白电泳检测。诱导表达过程如下:
1)活化菌种(37℃、12hr);
2)10%接种于LB培养基中(37℃、2hr);
3)加入诱导物IPTG,150rpm,18-22℃低温诱导4-20h;
4)离心收集菌体,加入10mM Tris·Cl(pH 8.0)悬浮;
5)破碎菌体(超声波破碎完全);
离心12,000rpm 10min 4℃;
收集上清及沉淀各10-15μL,分别电泳检测。
聚丙烯酰胺凝胶配置如下。
上样:上样10-15μl,电泳:130-150V恒压。
染色与脱色:电泳后取出凝胶,用蒸馏水冲洗后,放入染色液中,60rpm振荡染色1hr左右,脱色液中脱色2hr左右,脱色至凝胶背景透明,清水中漂洗至蛋白带清晰。
将重组质粒pEB-cry1Bb3,cry1Da4,cry1Id3和cry1Ja3,(见图6)转化到E.coli Rosetta(DE3)中,IPTG诱导表达,SDS-PAGE(12%)凝胶电泳。结果表明,cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因都能通过表达载体pEB在大肠杆菌中高效表达130kD蛋白,而经IPTG诱导的转入Rosetta(DE3)的pEB空载体没有特异的目的条带产生(见图5)。
将重组质粒pET21b-sip1A(见图6)转化到E.coli Rosetta(DE3)中,产生130kD蛋白,如图5。
3.2Bt菌株FH21及cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因编码蛋白的杀虫活性测定
将cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因表达蛋白,用水稀释到不同浓度,对鳞翅目害虫的杀虫活性,具体方法如下,采用饲料混合法进行杀虫生物活性测定。将制备好不同浓度梯度的表达蛋白样品并分装于经过消毒的培养皿中,分别与饲料搅拌混匀,挑选活跃的 初孵幼虫接于饲料上,每个处理重复3次,小菜蛾、棉铃虫、玉米螟农业害虫每个重复为30头试虫。阴性对照为10mmol/L Tris-Cl溶液作。试虫的饲养条件为相对湿度为70%-80%、温度为27℃的光照培养箱中培养,饲育48h后调查死、活虫数量,计算死亡率。采用POLO软件计算95%置信区间和LC50值。
表1 FH21菌株及表达蛋白对鳞翅目农业害虫杀虫活性测定结果
2014年Changlong Shu在期刊Applied and Environmental Microbiology发表了Cry1Bb2、Cry1Ja2对小菜蛾活性分别为0.32μg/mL(0.25–0.43μg/mL)、1.01μg/mL(0.72–1.38μg/mL)与测试结果相当,但对其它害虫没有活性,与本申请最接近的Cry1Ja1没有查到活性生测数据,而本申请中的Cry1Bb3、Cry1Ja3的对玉米螟还有活性,且Cry1Ja3对玉米螟的活性较高,Hernandez-Martinez 2008年发表在J.Invertebrate Path文章中指出cry1Da1对甜菜夜蛾9.5μg/g(3.2-18),对小菜蛾活性104μg/g(65-162),但本申请中Cry1Da3蛋白对小菜蛾的杀灭活性较高,同时对棉铃虫也有一定的杀灭作用;Cry1Id1相关基因没有发现其活性生测数据,本申请中的Cry1Id3对棉铃虫和小菜蛾具有较高的杀灭活性。本申请的基因拓宽了相关基因的杀虫谱。
本发明的有益效果:本发明分离克隆的Bt cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因序列及其基因表达产物能够对鳞翅目产生毒力,特别对于小菜蛾、棉铃虫、玉米螟、甜菜夜蛾,可扩大对鳞翅目害虫的杀虫谱,通过应用于转化微生物和植物,使它们表现出对相关害虫的毒性,可克服或延缓昆虫对工程菌和转基因植物抗药性的产生。
Claims (10)
1.苏云金芽胞杆菌菌株FH21,其保藏编号为:CGMCC No.10090。
2.苏云金芽胞杆菌菌株FH21在杀灭鳞翅目农业害虫中的应用。
3.杀虫蛋白Cry1Bb3,Cry1Da4,Cry1Id3或Cry1Ja3,其氨基酸序列分别如SEQ ID NO 2、SEQID NO 4、SEQ ID NO 6、SEQ ID NO 8所示。
4.cry1Bb3,cry1Da4,cry1Id3或cry1Ja3基因,分别编码杀虫蛋白Cry1Bb3,Cry1Da4,Cry1Id3或Cry1Ja3。
5.权利要求4所述的cry1Bb3,cry1Da4,cry1Id3或cry1Ja3基因,其核苷酸序列分别如SEQ IDNO1、SEQ ID NO 3、SEQ ID NO5、SEQ ID NO 7所示。
6.一种表达载体,其特征是含有权利要求4或5所述的cry1Bb3,cry1Da4,cry1Id3或cry1Ja3基因。
7.权利要求6所述的表达载体为pEB-cry1Bb3,pEB-cry1Da4,pEB-cry1Id3和pEB-cry1Ja3,其骨架载体为pEB,其结构如图6所示;
8.一种微生物转化体,其特征是含有权利要求4或5所述的cry1Bb3,cry1Da4,cry1Id3或cry1Ja3基因。
9.权利要求4或5所述的cry1Bb3,cry1Da4,cry1Id3或cry1Ja3基因在转基因物种杀灭小菜蛾,棉铃虫,玉米螟等农业害虫中的应用。
10.权利要求9所述应用,是将权利要求4或5所述的cry1Bb3,cry1Da4,cry1Id3和cry1Ja3基因表达的蛋白作为生物杀虫剂的有效成分。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410798930.6A CN104673706B (zh) | 2014-12-20 | 2014-12-20 | 苏云金芽胞杆菌fh21、杀虫基因,表达蛋白及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410798930.6A CN104673706B (zh) | 2014-12-20 | 2014-12-20 | 苏云金芽胞杆菌fh21、杀虫基因,表达蛋白及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN104673706A true CN104673706A (zh) | 2015-06-03 |
CN104673706B CN104673706B (zh) | 2018-03-06 |
Family
ID=53309300
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201410798930.6A Active CN104673706B (zh) | 2014-12-20 | 2014-12-20 | 苏云金芽胞杆菌fh21、杀虫基因,表达蛋白及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN104673706B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110022676A (zh) * | 2017-01-04 | 2019-07-16 | 先正达参股股份有限公司 | 用于控制植物有害生物的组合物和方法 |
CN110093301A (zh) * | 2019-05-30 | 2019-08-06 | 长江师范学院 | 一种苏云金芽孢杆菌及其在防治鳞翅目类害虫中的应用 |
US10480008B2 (en) | 2014-10-16 | 2019-11-19 | Poineer Hi-Bred International, Inc. | Insecticidal polypeptides having broad spectrum activity and uses thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4797276A (en) * | 1985-03-22 | 1989-01-10 | Mycogen Corporation | Control of cotton boll weevil, alfalfa weevil, and corn rootworm via contact with a strain of Bacillus thuringiensis |
CN101984045A (zh) * | 2010-09-29 | 2011-03-09 | 东北农业大学 | 苏云金芽孢杆菌cry8Nal基因,表达蛋白及其应用 |
-
2014
- 2014-12-20 CN CN201410798930.6A patent/CN104673706B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4797276A (en) * | 1985-03-22 | 1989-01-10 | Mycogen Corporation | Control of cotton boll weevil, alfalfa weevil, and corn rootworm via contact with a strain of Bacillus thuringiensis |
CN101984045A (zh) * | 2010-09-29 | 2011-03-09 | 东北农业大学 | 苏云金芽孢杆菌cry8Nal基因,表达蛋白及其应用 |
Non-Patent Citations (4)
Title |
---|
UNIPROT: "UniProtKB-V9I1J4(V9I1J4_BACTU)", 《UNIPROT》 * |
WILLIAM P. DONOVAN等: "Discovery and characterization of Sip1A: a novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae", 《APPL MICROBIOL BIOTECHNOL》 * |
孙晓东 等: "桂林喀斯特地貌分布区苏云金芽抱杆菌菌株的分离鉴定", 《中国生物防治》 * |
雷会霄: "苏云金芽孢杆菌的分离鉴定及杀虫基因的克隆", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10480008B2 (en) | 2014-10-16 | 2019-11-19 | Poineer Hi-Bred International, Inc. | Insecticidal polypeptides having broad spectrum activity and uses thereof |
CN110022676A (zh) * | 2017-01-04 | 2019-07-16 | 先正达参股股份有限公司 | 用于控制植物有害生物的组合物和方法 |
EP3565404A4 (en) * | 2017-01-04 | 2021-01-20 | Syngenta Participations AG | COMPOSITIONS AND METHODS FOR CONTROLLING PHYTO-PAVERS |
US11359209B2 (en) | 2017-01-04 | 2022-06-14 | Syngenta Participations Ag | Compositions and methods for control of insect pests |
CN110022676B (zh) * | 2017-01-04 | 2023-07-25 | 先正达参股股份有限公司 | 用于控制植物有害生物的组合物和方法 |
CN110093301A (zh) * | 2019-05-30 | 2019-08-06 | 长江师范学院 | 一种苏云金芽孢杆菌及其在防治鳞翅目类害虫中的应用 |
Also Published As
Publication number | Publication date |
---|---|
CN104673706B (zh) | 2018-03-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104946668A (zh) | cry1Ia基因及应用、由其编码的Cry1Ia蛋白及制备方法和应用 | |
CN102559554B (zh) | 苏云金芽胞杆菌cry1Ca基因,表达蛋白及其应用 | |
CN101984045B (zh) | 苏云金芽孢杆菌cry8Na1基因,表达蛋白及其应用 | |
CN103204912B (zh) | 对鳞翅目害虫高毒力杀虫基因cry2Ah-like及应用 | |
CN104388349B (zh) | 苏云金芽胞杆菌分泌杀虫基因sip1A,表达蛋白及其应用 | |
CN104611260B (zh) | 苏云金芽胞杆菌LTS290、杀虫基因cry57Ab、表达蛋白及其应用 | |
CN104673706B (zh) | 苏云金芽胞杆菌fh21、杀虫基因,表达蛋白及其应用 | |
CN105367634B (zh) | 一种Bt蛋白Cry1Ie5、其编码基因及应用 | |
CN105367633B (zh) | 一种BT蛋白CRY2Ab32、其编码基因及应用 | |
Zothansanga et al. | Diversity and toxicity of Bacillus thuringiensis from shifting cultivation (jhum) habitat | |
CN109929015B (zh) | 苏云金芽胞杆菌杀虫基因cry79Aa1、表达蛋白及其应用 | |
CN103333230B (zh) | 苏云金芽孢杆菌基因cry1Da3及其应用 | |
CN101926365B (zh) | 苏云金芽孢杆菌cry1Ai在抗虫中的用途、改造的mcry1Ai基因及应用 | |
CN101717437B (zh) | 苏云金芽孢杆菌Cry9E基因,蛋白及其应用 | |
CN104211790B (zh) | 一种高效杀灭同翅目害虫的Bt蛋白Cry21NJ、编码基因及其应用 | |
CN103570811B (zh) | 苏云金芽孢杆菌基因cry1Ah3及其应用 | |
CN105367636B (zh) | 一种Bt蛋白Cry1Dd1、其编码基因及应用 | |
CN103525837B (zh) | Bt蛋白Cry72Aa1操纵子基因及其应用 | |
CN102408475B (zh) | 一种Bt蛋白Cyt1Da1、其编码基因及应用 | |
CN103525835B (zh) | 一种Bt cry71Aa1基因及其编码蛋白和应用 | |
CN103525836B (zh) | 一种Bt Cry71Aa1操纵子基因及其编码蛋白和应用 | |
CN106011013A (zh) | 苏云金芽胞杆菌3-1-a、杀虫基因cry8Ax表达蛋白及其应用 | |
CN101413007B (zh) | 对鞘翅目害虫高效的苏云金芽胞杆菌cry8Ⅰ基因、蛋白及其应用 | |
CN102603876B (zh) | 一种Bt蛋白Cry59Ba1、其编码基因及应用 | |
CN103524605B (zh) | 一种Bt蛋白Cry72Aa1、其编码基因及应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |